This invention relates to a method for the selective adsorption of albumin from mixtures containing same and for substantially quantitative elution of the albumin by the use of a new series of dye-insoluble support compounds.
Albumin is a major protein in blood plasma or serum and is a commercially important product used in the medical field. The well known Cohn process or modifications thereof is generally employed in the fractionation of plasma into a number of medically useful and important protein fractions in addition to the production of albumin. Albumin, being one of the most soluble proteins, is among the last to be isolated in this long and tedious fractionation procedure for obtaining a variety of different plasma proteins. As a result, other methods have been sought for separating albumin from plasma, without sacrificing or being detrimental to the remaining proteins, which would be faster and more economical. In addition, it is frequently desired to isolate specific plasma components such as enzymes which are free of the major contaminant, albumin.
Recently a method has been described which indicated albumin in plasma could be selectively adsorbed onto and eluted from insoluble compounds which are made from a specific class of dyes coupled to agarose (German Offenlegungsschrift 2443119 filed Sept. 9, 1974; J. Travis and R. Pannell, Clin. Chim. Acta 49, pp. 49-52, 1973). This class of dyes is characterized by each dye having a triazine nucleus which is substituted with sulfonated aniline or naphthylamine groups. One of the dye-agarose compounds described is Sepharose 4B which is first activated with cyanogen bromide and then coupled to Blue Dextran. Blue Dextran is Cibacron Blue F3G-A in which the chlorine atom on the triazine moiety has been replaced with O-Dextran. The Sepharose 4B-Blue Dextran compound is reported to adsorb 96% of the albumin from plasma with substantially no adsorption of the other protein components. The albumin was about 80% eluted using a phosphate-ethanol eluting solvent at pH 2.4. A number of other dye-agarose compounds are disclosed including Cibacron Blue F3G-A coupled to cross-linked Sepharose 4B. Although a number of solutes are disclosed for eluting albumin, including a mixture of octanoic acid, Tris hydrochloride and sodium chloride, calcium, magnesium or potassium chloride with Tris hydrochloride, and high molar concentrations of aqueous area, no details are given on the efficiency of elution.
Although this method of Travis et al appears useful for selectively removing albumin from mixtures of proteins, the recovery of albumin is not sufficiently high to warrant the use of this process on a commercial scale. In addition, there is no indication as to the effectiveness in selective adsorption of albumin from protein solutions other than from plasma if the solutions contain higher or lower percentages of albumin as in certain Cohn fractions, e.g. crude gamma globulin, Cohn Fractions IV-1, IV, IV-4, the protein in Supernatant II + III, and Plasmanate.RTM. (plasma protein fraction isolated from Cohn Supernatant IV-1).